Legionella pneumophila is the etiologic agent of Legionnaires' disease, and is a facultative intracellularparasite of human macrophages and fresh water protozoa. A genetic analysis of L.pneumophila has been underway in order to identify bacterial factors that are necessary for intracellular infection and virulence. Recently, using site-specific mutagenesis, it was determined that the mip gene of L. pneumophila which encodes a 24,000 dalton surface protein (Mip) is required for the initiation of macrophage infection and for full virulence in animals. This proposal will examine mip at several new levels. We will determine whether this gene is critical for macrophage infection by other Legionella species. First, the distribution, expression, and structure of mip-like genes in the other species of Legionella will be determined. Then, the role of these mip-like genes in intracellular infection will be assessed by constructing mutants. A second aim of this proposal will employ genetic tools such as transcriptional gene fusions to determine more precisely the relationship between the level of mip expression and the level of macrophage infectivity. Thirdly, the regulation of mip will be studied under extra- and intracellular conditions. Attempts will be made to determine the molecular level at which mip is regulated, and to identify the gene(s) that regulates mip. From these studies, it may be possible to identify factors of L. pneumophila that are coordinately regulated with mip; some of these may prove to be "new" virulence factors. These studies will lend new insight into the molecular pathogenesis of the Legionellae and into the natural history of Legionnaires' disease. In addition, the knowledge gained here may help in understanding the biology of macrophages, other intracellular parasites, and other respiratory pathogens.